Antifungal antibiotic cepacidine A

ABSTRACT

Novel antifungal antibiotics Cepacidine A (A 1  and A 2  ),a novel microorganism Pseudomonas cepacia AF 2001 producing the same, and a process for producing the said antibiotics are disclosed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel antifungal antibiotics Cepacidine A(A₁ and A₂), a novel microorganism Pseudomonas cepacia AF 2001 producing the same, and a process for producing the said antibiotics.

2. Description of the Prior Art

As a variety of antifungal agents such as griseofulvin, nystatin, amphotericin-B and the like were separated from actinomycetes, chemotherapies for the treatment of fungal infections have been established. Another group of antifungal agents such as blasticidin, kasugamycin, polyoxin and validamycin was separated from actinomycetes and has been utilized in controlling fungal infections of plants. Thereafter, many antifungal substances were not only separated from various microorganisms occurring in nature but also produced by synthetic, chemical procedures. However, due to their poor efficacy or toxicity, most antifugal agents have not been utilized in commerce.

Antifungal agents in practical use do not function in a broad spectrum of activities nor do they exhibit a safe level of toxicity. These problems make it difficult to remedy deep antifungal infections. Moreover, since these antifungal agents do not have enough activities to kill fungi completely, rather having fungistatic activities, the use of those antifungal agents requires a long period of therapy. Therefore, there exists a need to develop novel antifungal agents having low toxicity, quick efficacy and fungicidal activity.

SUMMARY OF THE INVENTION

Cultivation of the novel microorganism Pseudomonas cepacia AF 2001 yields novel antibiotic substances Cepacidine A having powerful activities Against various yeasts and fungi, which have the formula: ##STR1## wherein R is H or OH. Where R is H, Cepacidine A₂ was designated; and where R is OH. Cepacidine A₁ was designated.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ultraviolet spectrum of Cepacidine A.

FIG. 2 shows the infrared spectrum of Cepacidine A in potassium bromide.

FIG. 3 shows the fast atom bombardment mass spectrum of Cepacidine A.

FIG. 4 shows the 400 MHz ¹ H NMR spectrum of Cepacidine A in deuterated dimethylsulfoxide.

FIG. 5 shows 100 MHz ¹³ C NMR spectrum of Cepacidine A in deuterated dimethylsulfoxide.

FIG. 6 shows the pyrolyzed gas chromatograms of Cepacidine A, asparagine and aspartic acid.

DETAILED DESCRIPTION OF THE INVENTION The Microorganism

Pseudomonas cepacia AF 2001 producing novel antifungal Cepacidine A was isolated from the soil samples collected in Munchon, Kyunggi-Do, Korea. The strain AF2001 was deposited at the Korean Federation of Culture Collections, Seoul, Korea, under the accession number KFCC 10773. The strain AF 2001 is a Gram-negative rod sized 0.4-0.6×1.0 -1.3 μm and motile by means of a polar flagella. It is oxidative and cytochrome positive. These characteristics serve to identify the strain as a Pseudemonas. A summary of the key characteristics and the carbon utilization pattern for the growth is shown in Table 1 below. The strain AF 2001 has similar characteristics to Pseudemonas cepacia disclosed in Bergy's manual of systematic bacteriology, 1984. However, the strain AF 2001 is different from Pseudomonas cepacia with regard to maltose, sucrose and adonitol utilization. It can grow on maltose and sucrose as a sole carbon source but can not grow on adonitol. In these aspects, the strain AF 2001 is thought to be a new Pseudomonas cepacia.

                  TABLE 1                                                          ______________________________________                                         Characteristics of Pseudomonas cepacia AF 2001                                 Cell type     Short rod                                                        Gram stain    Negative                                                         Biochemical characteristics:                                                   Oxidative     Positive                                                         Oxidase       Positive                                                         Catalase      Positive                                                         Urease        Negative                                                         DNase         Negative                                                         Lysine decarboxylase                                                                         Negative                                                         Nitrate reduction                                                                            Negative                                                         Poly β-hydroxybutyrate                                                                  Positive                                                         accumulation                                                                   Fluorescence  Negative                                                         Diffusible pigment                                                                           Positive                                                         Esculin hydrolysis                                                                           Positive                                                         Benzene ring cleavage                                                                        Ortho                                                            Growth at 4° C.                                                                       Negative                                                         Growth at 41° C.                                                                      Positive                                                         Growth at pH 3                                                                               Negative                                                         Growth at pH 9                                                                               Positive                                                         Carbon utilization:                                                            Positive:     maltose, sucrose, L-arabinose, mannose,                                        xylose, fructose, tartarate, dulcitol, salicin,                                cellobiose, fumaric acid, caprate, threonine,                                  lysine, arginine                                                 Negative:     adonitol, D-raffinose, lactose, maleic acid,                                   inulin, ethylene glycol, phthalic acid,                                        L-isoleucine, starch, melibiose                                  ______________________________________                                    

Production of the Antibiotic

Cultures of Pseudomonas cepacia AF 2001 were maintained at -85° C . When needed, working stock cultures were prepared on agar slants composed of glucose 3%, peptone 1.5% and agar 2%. The slants were incubated at 28°C. for 24 hours and used to inoculate germinator flasks containing 100 ml of medium in 500 ml Erlenmeyer flasks. This medium consisted of bactopeptone 1%, yeast extract 1% and glucose 3% The germinator was incubated for 24 hours at 28°C. on a rotary shaker at 300 rpm, and then used to inoculate(1%) the same medium(10 L). The fermentation was run for 72 hours at 28°C. with an agitation rate of 300 rpm. Progress of the fermentation and the subsequent isolation steps were monitored by paper-disc, agar diffusion assay with Candida albicans ATCC 38245 as the assay microorganism. Cepacidine A was isolated and purified by the procedure outlined in the diagram below. The fermentation broth(10 L) was mixed with an equal volume of isopropanol(10 L), and the mixture was adjusted to pH 4.0 with concentrated HCl. The mixture was centrifuged and the cell pellet was discarded. The supernatant was then concentrated under reduced pressure to remove isopropanol. The precipitate that formed during the concentration process contained the bioactivity, as shown by conventional agar diffusion assay. The concentrate and accompanying precipitate were stored at 4° C. for 6 hours to allow the precipitation to proceed to completion. The precipitate was collected by filtration with diatomaceous earth, washed with water and then eluted with 50% isopropanol in water. This solution was concentrated in vaccum to remove isopropanol. After extraction of this concentrate with isobutanol-methanol (8:2), the solvent extract was then concentrated under reduced pressure. The precipitate that formed during the concentration was collected by centrifugation and dissolved in 50% isopropanol adjusted to pH 10.0 with NaOH. This solution was diluted with 10 volume distilled water and applied to diaion HP-20 resin column chromatography. After elution with 50% isopropanol, the eluent was concentrated in vaccum to a small volume. The precipitate formed during the concentration was dissolved in 50% isopropanol and applied to alumina column chromatography. The passthrough that contained the bioactivity was concentrated to a small volume and stored at 4° C. for 24 hours to allow precipitation. After washing with water, the precipitate was dissolved in 50% isopropanol and applied on a preparative ODS-silica gel column of Waters μ-Bondapak C18 (3×30 cm, 10 μm) and developed with acetonitrile-water (6:4, pH 3.5). The fraction containing anti-candidal activity was collected and concentrated in vaccum to give residues of pure Cepacidine A (100 mg). Cepacidine A was further resolved into two closely related components, i.e., A₁ and A₂ by HPTLC on silica gel(Merck Silica gel 60 F₂₅₄), eluting with isopropanol-conc. ammonia-water(4:2:1). Visualization of the components was accomplished either by charring after spraying with a solution containing 5% ammonium molibdate and 0.1% ceric ammonium sulfate in 10% sulfuric acid or by spraying conventional ninhydrin reagent. The Rf values of A₁ and A₂ are 0.53 and 0.58, respectively. The producing ratio of A₁ and A₂ was determined to be 9:1 according to the quantitative analysis of the amounts of purified A₁ and A₂. Unfortunately, it was very difficult to isolate a large amount of pure A₁ and A₂, because Cepacidine A was not separated into A₁ and A₂ using the HPLC system described above. Therefore, the mixture of A.sub. and A₂ was used for the following studies, i.e., biological activity, physicochemical properties and structural elucidation.

Diagram on Isolation Procedures of Cepacidine A₁ and A₂ ##STR2## Biological Activity

The in vitro antifungal activities of Cepacidine A and amphotericin B against a broad range of medically important fungi are listed in Table 2. The activities were determined by the twofold agar dilution method on potato dextrose agar medium. Cepacidine A exhibited a broad antifungal spectrum against all strains tested. Particularly, Cepacidine A was highly active against dermatophytes, namely Microsporum canis, Trichophyton spp, and Epidermophyton spp., and true yeast at concentrations lower than 0.049 μml. The activities of Cepacidine A were greater than those of amphotericin B in most strains. However, no antibacterial activity was detected (MIC>100 μg/ml) when Cepacidine A was assayed against the bacteria i.e., Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.

                  TABLE 2                                                          ______________________________________                                         Antifungal spectrum of                                                         Cepacidine A and Amphotericine-B                                                                    MIC (μg/ml)                                                                       Cepaci-  Amphoter-                                  organism        KCTC No*   dine A** icin-B                                     ______________________________________                                         Candida albicans                                                                               1940       0.391    0.782                                      Candida albicans                                                                               38245      0.391    0.782                                      Candida glabrata                                                                               1714       0.013    0.196                                      Cryptococcus neoformans                                                                        1197       0.025    0.098                                      Saccharomyces cerevisiae                                                                       1213       0.049    0.098                                      Aspergillus niger                                                                              2119       0.098    0.196                                      Microsporum gypseum                                                                            1252       0.196    0.196                                      Microsporum canis                                                                              11621      0.025    0.391                                      Epidennophyton floccosum                                                                       1246       0.049    3.125                                      Trichophyton mentagrophyte                                                                     6085       0.049    3.125                                      Trichophyton rubrum                                                                            38484      0.049    0.782                                      Fusarium oxysporum                                                                             6084       0.196    0.196                                      Rhizopus stolonifer                                                                            6062       0.391    0.391                                      ______________________________________                                          *:Korean Collection for Type Cultures                                          **:Two component mixture                                                 

Physico-chemical Properties

The physico-chemical properties of Cepacidine A are summarized in Table 3 below. Cepacidine A was isolated as white powder. The melting point ranged between 210° C. and 214° C. Cepacidine A is insoluble in ethyl acetate, hexane, ether and benzene, and is hardly soluble in water, methanol, ethanol, isopropanol, butanol and acetone, while it is soluble in DMSO, alkali aqueous solution and acidic aqueous solution. A 50% aqueous solution of alcohol increases solubility. Cepacidine A showed positive color reactions to aniline and ninhydrin reagents. The Rf value of Cepacidine A on silica gel TLC developed with n-butanol-acetic acid-water(3:1:1) was 0.18. However, the Rf values of Cepacidine A on silica gel TLC developed with isopropanol-water-saturated aqueous ammonia(4:1:2) were separated as 0.53 and 0.58 so that the compound with the Rf value of 0.53 was named Cepacidine A₁, and that with 0.58, Cepacidine A₂. Since Cepacidine A₁ and Cepacidine A₂ were hardly obtained separately by prep-HPLC, unfortunately, the mixture, Cepacidine A, was used for all spectrometric analyses, except TLC and amino acid analysis by HPLC. The mixture has a 9:1 ratio of Cepacidine A₁ and Cepacidine A₂. The UV spectrum of Cepacidine A dissolved in water showed two maximum absorption peaks at 232 nm and 274 nm, and the spectrum in DMSO showed only one peak at 278 nm. Cepacidine A is very stable in an aqueous solution between pH 2 and pH 11, while unstable in an aqueous solution above pH 11.5 so that Cepacidine A loses antifungal activities readily. The molecular formula of Cepacidine A₁ was determined to be C₅₂ H₈₅ O₂₂ N₁₁ by HRFAB-MS, ¹³ C NMR and elemental analysis (calcd : C 51.4, H 7.0, N 12.7, 0 29.0; found : C 51.5, H 8.0, N 11.0, 0 29.5), Cepacidine A₂, C₅₂ H₈₅ O₂₁ N₁₁. The molecular ions of Cepacidine A₁ and Cepacidine A₂ by HRFAB-MS were shown at m/z 1216.5999(MH⁺, calcd: 1216.5949) and 1200.5978(MH⁺, calcd.:1200.5999), respectively.

                  TABLE 3                                                          ______________________________________                                         Physico-Chemical Properties of Cepacidine A                                                 Cepacidine A.sub.1                                                                        Cepacidine A.sub.2                                     ______________________________________                                         appearance     white powders                                                                               white powders                                      MP             210-214° C.                                                                          210-214° C.                                 UV λ.sub.max nm (log e) in H.sub.2 O                                                   232 (2.8), 274 (1.7)                                                                        232 (2.8), 274 (1.7)                               in DMSO        278 (1.2)    278 (1.2)                                          IR (KBr) γ.sub.max cm.sup.-1                                                            3352, 2924, 2854,                                                                           3352, 2924, 2854,                                                 1666, 1539   1666, 1539                                                        1412, 1252, 1069,                                                                           1412, 1252, 1069,                                                 557          557                                                 α!.sub.D.sup.25 H.sub.2 O                                                              +20.8        +20.8                                              TLC Rf value (n-BuOH:                                                                         0.18         0.18                                               AcOH:H.sub.2 O = 3:1:1)                                                        (iso-PrOH:H.sub.2 O:said                                                                      0.53         0.58                                               NH.sub.4 OH = 4:1:2)                                                           molecular formula                                                                             C.sub.52 H.sub.85 O.sub.22 N.sub.11                                                         C.sub.52 H.sub.85 O.sub.21 N.sub.11                HRFAB-MS (M + H).sup.+  calcd                                                                 1216.5949    1200.5999                                          found          1216.5999    1200.5978                                          ______________________________________                                    

Structure Elucidation

Since Cepacidine A showed positive color reaction to ninhydrin reagent, amino acid analysis was carried out by TLC and HPLC after acid hydrolysis. The analysis revealed Cepacidine A₁ consists of β-hydroxy Asx, Asx, Ser, Gly and 2,4-diaminobutyric acid(1:1:2:1:1), and Cepacidine A₂, Asx, Ser, Gly and 2,4-diaminobutyric acid(2:2:1:1). For amino acid analysis, the solution obtained from acid hydrolysis was eluted through octadecyl column. The remnant inside the column was washed with 50% isopropanol and collected for NMR experiments. The inspection of NMR experiments revealed the remnant is a type of β-amino acid with 18 carbons. This amino acid is a long chain of carbons with 3 functional groups and 1 methyl group, which were determined to be 1 primary amine and 2 hydroxyl groups by ¹ H NMR, ¹³ C NMR, COSY, HETCOR, NOESY, HOHAHA and HMBC. Because Cepacidine A showed positive color reaction to aniline reagent, saccharide analysis was carried out by cellulose TLC and HPLC after acid hydrolysis. The analysis revealed both Cepacidine A₁ and Cepacidine A₂ include xylose.

The spectrum of Cepacidine A obtained from low resolution FAB-MS shows only MH⁺ ions of Cepacidine A₁ (m/z 1216) and Cepacidine A₂ (m/z 1200) except a few small fragments and xylose fragment. This phenomenon suggests Cepacidine A can be a cyclic peptide. The NMR experiments such as NOESY, HOHAHA and HMBC clarified this suggestion.

The sum of the calculated number of carbons of components obtained from amino acid analysis and saccharide analysis, and the number of carbons of C18 long chain amino acid is only 43. However, the ¹³ C NMR spectrum gives 50 peaks. Therefore, the presence of the other components can be considered. The ¹ H NMR spectrum of Cepacidine A reveals the presence of aromatic ring. Since the four carbon signals at 114.70, 137.00, 132.20 and 156.50 ppm are characteristic peaks caused by para-hydroxy phenyl group, the presence of Tyr can be expected, but amino acid analysis with HPLC does not show the peak of Tyr so that the presence of a derivative of Tyr can be considered. In the COSY spectrum, cross peaks among 4.19, 5.06, 6.67 and 7.14 ppm are observed. In addition, HETCOR shows four correlated peaks such as 4.19/60.40, 5.06/70.96, 6.67/114.70 and 7.14/127.00(¹ H NMR/¹³ C NMR). These phenomena suggest one of β-protons of Tyr is substituted with a hydroxyl group. In order to clarify this, a chemical experiment was carried out. The UV spectrum of Cepacidine A in DMSO(FIG. 1) shows one peak at 278 nm. An addition of TFA into Cepacidine A caused bathochromic shift of λmax to 312 nm. An elimination of α-proton and β-proton of Tyr can cause a conjugation. Then, two carbon peaks at 114.70 and 127.00 in the ¹³ C NMR spectrum must be ε and δ carbons of Tyr and denote two carbon intensity each. As a result, the number of carbon in Cepacidine A is not 50 shown in the ¹³ C NMR spectrum but 52.

As mentioned before, molecular formula of Cepacidine A.sub. and Cepacidine A₂ was determined to be C₅₂ H₈₅ O₂₂ N₁₁ and C₅₂ H₈₅ O₂₁ N₁₁ by HRFAB-MS and elemental analysis. In order to determine Asx, pyrolyzed GC was carried out. Chromatograms of Asn and Asp as references are shown in FIG. 6. Two chromatograns can be distinguished by the characteristic peaks at retention time=47 min. Since the peaks of Cepacidine A at the same retention time are same as those of Asp, Asx contained in Cepacidine A must be Asp. NOESY and HOHAHA experiments have revealed xylose is connected to one of hydroxyl groups of C18 long chain amino acid. Here, if one counts all elements contained in the determined components of Cepacidine A₁ , only β-hydroxy Asx is not counted. Until now Gly (1), Ser (2), Asp (1), β-hydroxy Tyr (1), 2,4-diaminobutyric acid (1), xylose (1), and C18 amino acid(C₁₈ H₃₈ O₄ N₂) were determined and the sum of elements contained in those components are C₄₈ H₉₅ O₂₇ N₉. However, because Cepacidine A is a cyclic peptide and xylose is connected to C18 amino acid, the formula must be C₄₈ H₇₉ O₁₉ N₉. The difference between this formula and that obtained from HRFAB-MS gives C₄ H₆ O₃ N₂ for Cepacidine A₁. Therefore, the undetermined components, β-hydroxy Asx of Cepacidine A₁ must be β-hydroxy Asn, and Asx of Cepacidine A₂, Asn, respectively. The components of Cepacidine A₁ and Cepacidine A₂ are listed in Table 4.

                  TABLE 4                                                          ______________________________________                                         Components of Cepacidine A.sub.1 and A.sub.2                                   components      Cepacidine A.sub.1                                                                        Cepacidine A.sub.2                                  ______________________________________                                         Glycine         1          2                                                   Serine          2          2                                                   Aspartic acid   1          1                                                   Asparagine      0          1                                                   β-Hydroxyasparagine                                                                       1          1                                                   2,4-Diaminobutyric acid                                                                        1          1                                                   β-Hydroxytyrosine                                                                         1          1                                                   C.sub.18 Amino acid                                                                            1          1                                                   Xylose          1          1                                                   ______________________________________                                    

Experimental

FAB-MS was measured on a Jeol DX 303 spectrometer. UV and IR were recorded on a Beckman DU-70, and on a Bruker IFS 66, respectively. NMR spectra were recorded on a Bruker ARX 400 spectrometer in DMSO-d₆ and 95% DMSO-d₆ /5% D₂ 0. The elemental analysis data were obtained on Foss Heraeus CHN-0-Rapid. TLC was performed on pre-coated Silica gel plates(Merck catalog No. 5642). Pyrolysed GC was measured on a Schimadzu GC 15A and a JHP-35 Pyrolyser with a CBP-5 column. For amino acid analysis Cepacidine A was hydrolyzed with 6N HCl at 105° C. for 8 hr. and Waters amino acid conversion kit and Waters amino acid analysis column were used, Waters Fluorescence 420, as a detector. For saccharide analysis, Cepacidine A was hydrolyzed with 10% H₂ SO₄ at 100°C. for 1 hr, and Waters carbohydrate analysis system and Waters carbohydrate column were used, Waters R1410, as a detector. 

What is claimed is:
 1. An antifungal compound having the following formula: ##STR3## wherein R is H or OH.
 2. An isolated microorganism Pseudomonas sp. AF
 2001. 3. A process for producing the antifungal antibiotics according to claim 1, which process comprises fermentation of a microorganism Pseudomonas sp. AF 2001, and extracting and purfying the fermentation broth of said strain to obtain said antibiotics. 